We propose to elucidate the biochemical, genetic and nutritional factors that regulate de novo thymidylate (dTMP) synthesis at the sites of DNA synthesis, and establish the mechanisms underlying impairments in this pathway. Impaired folate- and vitamin B12-dependent one-carbon (1C) metabolism is associated with numerous common pathologies, but mechanisms have yet to be established. Recently, we reported that the Shmt-/+ mouse model accumulates uracil in DNA and is sensitized to intestinal tumors and neural tube defects. SHMT1 is the only reported folate-dependent enzyme whose disruption results in folate-responsive neural tube defects. Our findings indicate that nuclear and mitochondrial dTMP biosynthesis underlies folate- and vitamin B12-related disease. As a result of these studies, our fundamental understanding of 1C pathways in the nucleus and mitochondria will be established, their regulation by specific vitamins and metabolites elucidated, and their role in maintaining genome integrity characterized. These studies will also determine if there are metabolic interactions between nuclear de novo dTMP biosynthesis and epigenetic modifications to DNA. The results will inform future human studies that translate into medical practice and public health policy for the prevention and treatment of folate- and vitamin B12-associated pathologies. The three related and overlapping areas to be investigated are: Aim I. Determine the contributions of the enzymes MTHFD1 and KDM1 to vitamin B12- and folate-mediated 1C metabolism in the nucleus. These studies will identify and characterize the role of two folate-utilizing enzymes, MTHFD1 and KDM1, as components of a nuclear multienzyme complex that synthesizes dTMP and associates with the DNA replication machinery. These studies will also determine if nuclear dTMP biosynthesis and epigenetic chromatin modifications are interdependent through their use of folate cofactors. Aim II. Elucidate the determinants and mechanisms of nuclear dTMP synthesis regulation. These studies will determine the role, regulation and contribution of nuclear folate-dependent enzymes in dTMP synthesis. Studies include investigations into the nutritional, metabolic and genetic factors that govern nuclear dTMP biosynthesis and genome stability. AIM III. Elucidate the determinants and mechanisms of mitochondrial dTMP synthesis regulation. These studies will determine the role, regulation and contribution of mitochondrial folate-dependent enzymes in dTMP synthesis, including the nutritional, metabolic and genetic factors that govern mitochondrial dTMP biosynthesis and mitochondrial genome stability. PUBLIC HEALTH RELEVANCE: Folate- and vitamin B12-associated pathologies are common but their underlying mechanisms and pathways are not understood, and folic acid supplementation alone is not sufficient to reduce or eliminate risk of these pathologies and may do harm. The experiments proposed in this application build on recent findings from our laboratory that several folate- and vitamin B12- related pathologies result from impaired thymidylate biosynthesis. The results from these experiments will inform future human studies and ultimately translate into medical practice and public health policy for the prevention and treatment of folate- and vitamin B12-associated pathologies.